- 學位論文
源自不同牛種卵母細胞質所產製複製牛耳朵細胞之抗熱及抗氧化能力
Thermotolerance and Antioxidative Capacity of Ear Fibroblasts Derived from Cloned Cattle with Different Ooplasm Origins
摘要
本研究室已利用體細胞核轉置(somatic cell nuclear transfer, SCNT)技術將荷蘭牛 (Holstein, H) 供核細胞 (donor cell, d) 搭配台灣黃牛 (Taiwan yellow cattle, Y) 或荷蘭牛卵母細胞質 (ooplasm, o) 而成功產製細胞質源自不同牛種之體細胞複製荷蘭牛 (cloned Holstein)。初步之結果雖顯示卵母細胞質源自台灣黃牛所產製複製荷蘭牛 (Yo-Hd) 之耳朵纖維母細胞的熱耐受性優於卵母細胞質源自荷蘭牛 (Ho-Hd) 所產製者，但複製荷蘭牛在熱耐受性及抗氧化能力之分子調控機制仍未清楚，且此等抗緊迫能力是否能傳承至後代亦需進一步探討。因此於本研究中將比較源自荷蘭牛、台灣黃牛、卵母細胞質源自荷蘭牛 所產製複製牛(Ho-Hd)及其後代(Ho-Hd-F1)、以及卵母細胞質源自台灣黃牛所產製複製牛(Yo-Hd)及其後代(Yo-Hd-F1)之耳朵纖維母細胞的熱耐受性及抗氧化能力差異。結果顯示，Yo-Hd及Yo-Hd-F1牛耳朵纖維母細胞之粒線體D-loop區的多態性與台灣黃牛相同，因此證實此等牛之細胞質源自台灣黃牛。進一步利用熱緊迫處理也發現，細胞質源自台灣黃牛之牛種 (包括Y、Yo-Hd及Yo-Hd-F1) ，其耳朵纖維母細胞之促凋亡現象，包括細胞凋亡率、Bax/Bcl-2比率、以及Caspases-3、Caspases-8、 Caspases-9蛋白質表現量均顯著 (P < 0.05) 低於細胞質源自荷蘭牛之牛種 (包括H、 Ho-Hd及Ho-Hd-F1)；反之，抑凋亡蛋白質HSP-70之表現量則以細胞質源自台灣黃牛之牛種顯著 (P < 0.05) 高於細胞質源自荷蘭牛之牛種者。而且，細胞質源自台灣黃牛之耳朵纖維母細胞經熱緊迫處理後之氧化磷酸化蛋白質 (oxidative phosphorylation proteins) 包括Complex III 與 Complex IV，以及抗氧化之glutathione peroxidase (GPx)蛋白質的表現量亦均顯著 (P < 0.05) 高於細胞質源自荷蘭牛之牛種者；而Yo-Hd及Yo-Hd-F1牛耳朵纖維母細胞經熱緊迫處理後之catalase (CAT)蛋白質的表現也顯著 (P < 0.05) 高於Ho-Hd-F1牛者。此外，細胞質源自台灣黃牛之耳朵纖維母細胞經H2O2處理後之細胞凋亡率顯著(P < 0.05)低於細胞質源自荷蘭牛之牛種；而GPx蛋白質的表現量則顯著 (P < 0.05) 高於細胞質源自荷蘭牛之牛種者。然而，Yo-Hd和Yo-Hd-F1牛耳朵纖維母細胞經H2O2處理後之 CAT蛋白質表現雖高於Ho-Hd和Ho-Hd-F1牛者，但各處理組別間無顯著(P > 0.05)差異。綜合上述之結果說明，母細胞質源自台灣黃牛所產製之複製荷蘭牛 (Yo-Hd) 及其後代 (Yo-Hd-F1) 之耳朵纖維母細胞的熱耐受性及抗氧化能力均優於卵母細胞質源自荷蘭牛所產製之複製荷蘭牛 (Ho-Hd) 及其後代 (Ho-Hd-F1).
並列摘要
We have previously produced cloned Holstein by using somatic cell nuclear transfer (SCNT) from reconstructed embryos containing ooplasm from different breeds. Results demonstrated that the ear fibroblasts derived from cloned Holstein reconstructed using the Taiwan native yellow cattle ooplasm (Yo) are more thermotolerant than ear fibroblasts derived from cloned Holstein reconstructed using the Holstein ooplasm (Ho). However, molecules mediating the thermotolerance and antioxidant ability of ear fibroblasts of these cloned Holstein remained unclear. Moreover, whether these traits are transmissible to their offspring requires further investigations. The objective of this study was to compare the thermotolerances and antioxidant abilities of ear fibroblasts derived from Holstein (H) and Taiwan native yellow cattle (Y), and cloned Holstein reconstructed with recipient ooplasm from different bovine breeds (Yo-Hd, Ho-Hd), and their offspring (Yo-Hd-F1, Ho-Hd-F1). Polymorphisms in mitochondrial DNA (mtDNA) D-loop of ear fibroblasts derived from Yo-Hd and Yo-Hd-F1 indicated that the cytoplasm is originated from Bos indicus (Y). After heat shock, the apoptotic rates, B-cell lymphoma 2-associated X protein/B-cell lymphoma 2 ratios (Bax/Bcl-2), and relative expression levels of cysteine-aspartic proteases (caspases)-3, -8, and -9 of ear fibroblasts with Y-originated cytoplasm (including Y, Yo-Hd, and Yo-Hd-F1) were all significantly (P < 0.05) lower than those of ear fibroblasts with H-originated cytoplasm (including H, Ho-Hd, and Ho-Hd-F1). In contrast, the relative expression level of HSP-70 was significantly (P < 0.05) higher in ear fibroblasts with Y-originated cytoplasm than that of ear fibroblasts with H-originated cytoplasm. Moreover, oxidative phosphorylation proteins (Complex III and IV) of ear fibroblasts with Y-originated cytoplasm were significantly (P < 0.05) greater than those of ear fibroblasts with H-originated cytoplasm. The catalase (CAT) expressions in heat-shocked ear fibroblasts derived from Yo-Hd and Yo-Hd-F1 cattle were significantly (P < 0.05) higher than those of fibroblasts derived from Ho-Hd-F1 cattle. The level of glutathione peroxidase (GPx) expressions were higher (P < 0.05) in ear fibroblasts with Y-originated cytoplasm compared to those of fibroblasts with H-originated cytoplasm. Furthermore, after exposure to H2O2, the apoptotic rate of ear fibroblasts with Y-originated cytoplasm was lower (P < 0.05) than that of cells with H-originated cytoplasm. The expression of glutathione peroxidase was higher (P < 0.05) in ear fibroblast cells with Y-originated cytoplasm compared to ear fibroblasts with H-originated cytoplasm. In addition, the expression of catalase in ear fibroblasts derived from Yo-Hd and Yo-Hd-F1 cattle was higher, but not statistically significant (P > 0.05) than that of cells derived from Ho-Hd and Ho-Hd-F1 cattle. In conclusion, the ear fibroblasts derived from cloned Holstein reconstructed using the Y ooplasm and Yo-Hd-F1 had better thermotolerances and antioxidant abilities than ear fibroblasts derived from cloned Holstein reconstructed with the H ooplasm and Ho-Hd-F1.